Non-fouling polymeric surface modification and signal amplification method for biomolecular detection

ABSTRACT

An article such as a biosensor having a nonfouling surface thereon is described. The article comprises: (a) a substrate having a surface portion; (b) a linking layer on the surface portion; (c) a polymer layer comprising brush molecules formed on the linking layer; and (d) optionally but preferably, a first member of a specific binding pair (e.g., a protein, peptide, antibody, nucleic acid, etc.) coupled to the brush molecules. The polymer layer is preferably formed by the process of surface-initiated polymerization (SIP) of monomeric units thereon. Preferably, each of the monomeric units comprises a monomer (for example, a vinyl monomer) core group having at least one protein-resistant head group coupled thereto, to thereby form the brush molecule on the surface portion. Methods of using the articles are also described.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/717,430; filed Sep. 15, 2005, the disclosure ofwhich is incorporated by reference herein in its entirety.

This application is related to Ashutosh Chilkoti and Hongwei Ma, Atunable nonfouling surface of oligoethylene glycol, U.S. patentapplication Ser. No. 10/783,054, filed Feb. 20, 2004 (Docket No.5405-318), the disclosure of which is incorporated by reference hereinin its entirety.

This invention was made with Government support under grant no. R01CI-00097 from the Centers for Disease Control/NCID. The United StatesGovernment has certain rights to this invention.

FIELD OF THE INVENTION

This invention relates to a non-fouling polymeric surface modificationand signal amplification method for ultra-sensitive biomoleculardetection.

BACKGROUND OF THE INVENTION

Glass and silicon oxide are widely used substrates for biosensors,clinical immunoassay diagnostics, and cell culture (Ratner, Schoen etal. 1996) and as solid supports for the synthesis of peptides,carbohydrates, and DNA (Seeberger and Haase 2000). The modification ofsilicon oxide to modulate protein and cell interactions has proven tochallenging for a number of technical reasons: 1) the formation ofsilane self-assembled monolayers (SAMs), the most common route tofunctionalize glass and other metal oxides, is complicated by thesensitivity of most silanes to humidity and their propensity to formpolymeric multi-layers (Wasserman, Tao et al. 1989; Ulman 1996). 2) Asis typical to most “grafting to” approaches, the passivation of siliconoxide by grafting polyethylene glycol (PEG) to the surface using silanechemistry (Emoto, Harris et al. 1996; Yang, Galloway et al. 1999) doesnot provide a high surface density of PEG due to the excluded volumeeffect (Knoll and Hermans 1983). Consequently, grafted PEG coatings onglass decrease the adsorption of proteins, but do not reduce theiradsorption below the nominal limit of several ng/cm² (Zhu, Jun et al.2001). Several approaches such as the sequential grafting of PEGs ofdifferent chain lengths (Nagasaki, Ishii et al. 2001) and cloud-pointgrafting of PEG (Kingshott, Thissen et al. 2002) have been taken tosolve this problem, but only with limited success. 3) It is alsodifficult to stamp silanes onto glass with the ease and reproducibilitywith which alkanethiols can be patterned by micro-contact printing andother soft lithography methods so that the patterning of PEG on to glassby soft lithography has only been marginally successful (Xia, Mrksich etal. 1995; StJohn and Craighead 1996).

For the detection of a variety of biological molecules such as protein,RNA, and DNA in complex biological fluids, the minimization ofnon-specific protein binding plays a very important in improving thedetection limit and sensitivity. The reduction of adsorption of proteinand other biomolecules is important for the development of interfacialsensors for two reasons: first, for the broad class of sensors that arelabel-free, i.e., in which the binding event is directly transduced asthe detected signal (e.g., surface plasmon resonance (SPR) spectroscopy,localized or nanoparticle-based surface plasmon resonance (nanoSPR),surface enhanced Raman scattering (SERS), ellipsometry, gravimetricsensors such as quartz-crystal microbalance dissipation (QCM-D) andsurface acoustic wave (SAW) sensors, etc.) reduction of proteinadsorption to ultra-low levels (<1 ng/sq. cm) is critical to generate ahigh signal-to-noise ratio (SNR) by reducing the noise due toadventitious adsorption. For the class of interfacial sensors that use alabel to generate the detected signal, the elimination of backgroundadsorption is similarly important to reduce noise. Finally, for theclass of sensors that incorporate an amplification step prior to orduring generation of the detected signal, the effective elimination ofadventitious adsorption or binding of biomolecules or other reagents iscritical, as adventitiously bound molecules can be amplified, so thatthe increase in signal (S) afforded by the amplification step is in manycases compromised by the concomitant amplification of the backgroundnoise (N), so that the gains in SNR are modest, at best.

The increasing technological push towards ultra-sensitive detection inbiomolecular arrays—DNA, protein and carbohydrate—similarly requiresextremely low background signals so that a high SNR can be attained (Zhuand Snyder 2003). However, most commercially available chemical surfacemodifications usually have high auto-fluorescence or non-specificbinding of reagents and analytes. This issue is increasingly crucialwhen the spot size of commonly used microarrays becomes smaller andsmaller, even down to the sub-micron length scale. Although some of thecurrent surface modification techniques work well for microarrays (Zhuand Snyder 2003), the routine use of micro- and nano-arrays forbiomolecules still poses substantial challenges in engineering adetection system that is capable of resisting non-specific adsorption ofbiomolecules down to the pg/cm² level and allows direct detection ofanalytes without elaborate and expensive amplification techniques.

SUMMARY OF THE INVENTION

A first aspect of the present invention is an article (preferably abiomolecular detector or biosensor such as a microarray) having anonfouling surface thereon, the article comprising:

(a) a substrate having a surface portion;

(b) a linking layer on the surface portion; and

(c) a polymer layer formed on the linking layer, preferably by theprocess of surface-initiated polymerization (SIP) of monomeric unitsthereon. Preferably, each of the monomeric units comprises a monomer(for example, a vinyl monomer) core group having at least oneprotein-resistant head group coupled thereto, to thereby form a brushmolecule on the surface portion. The brush molecule preferably comprisesa stem formed from the polymerization of the monomer core groups, and aplurality of branches foamed from the head group projecting from thestem; and

(d) optionally but preferably, a first member of a specific binding pair(e.g., a protein, peptide, antibody, nucleic acid, etc.) coupled to thebrush molecule.

In some embodiments, the member of a specific binding pair furthercomprises an extended nucleic acid conjugated thereto, the extendednucleic acid produced by the process of enzymatic extension withterminal transferase (TdTase).

In some embodiments, wherein the first member is a first nucleic acid,the detector optionally further comprising a second nucleic acid as asecond member of the binding pair hybridized to the first member. Someembodiments thereof further comprise an extended nucleic acid producedby the process of enzymatic extension with terminal transferase coupledto either the first or second nucleic acid.

In some embodiments, particularly where the first member of the bindingpair is a protein or peptide, the detector further comprises: a secondmember of the specific binding pair coupled to the first member of thespecific binding pair; an antibody specifically bound to the secondmember to form a sandwich, the antibody having a first nucleic acidconjugated thereto; and an extended nucleotide coupled to the firstnucleotide, the extended nucleic acid produced by the process ofenzymatic extension with terminal transferase.

A second aspect of the present invention is a method of making anarticle (preferably a biomolecular detector such as a microarray) havinga nonfouling surface thereon, the method comprising: (a) providing asubstrate having a surface portion; (b) depositing a linking layer onthe surface portion; and (c) forming a polymer layer on the linkinglayer by the process of surface-initiated polymerization of monomericunits thereon, with each of the monomeric units comprising a monomer(for example, a vinyl monomer) core group having at least oneprotein-resistant head group coupled thereto, to thereby form a brushmolecule on the surface portion; the brush molecule comprising a stemformed from the polymerization of the monomer core groups, and aplurality of branches formed from the hydrophilic head group projectingfrom the stem.

In some embodiments the polymer comprises a copolymer ofmethoxy-terminated OEGMA and hydroxy-terminated OEGMA. Such embodimentsmay further comprise the step of coupling a compound (e.g., a firstmember of a specific binding pair) having an amine group to the hydroxygroup via the amine group. In other embodiments the polymer comprises ofvinyl monomer bearing other head groups such as hydroxyl (OH), glycerol,or groups known in the art as kosmotropes (see, e.g., Kane et al.,infra).

In some embodiments the copolymer is synthesized on the surface portionby SI-ATRP, wherein the hydroxy groups are converted to COOH groups byany suitable reaction such as reaction with N-hydroxy succinimide (NHS),and wherein the amine groups are coupled to the COOH.

In some embodiments the polymer comprises a copolymer of OEGMA andsodium methacrylate, the copolymer having sodium carboxylate groups. Insome embodiments the sodium carboxylate group is converted to a COOHgroup by reaction with an acid such as HCl, and a protein or peptide iscoupled to the COOH groups via their N-terminal amine or lysine residues(by any suitable reaction such as NHS/EDC coupling).

In some embodiments the polymer is OEGMA, the OEGMA containing ofthioester groups; and wherein the member of a specific binding pair iscoupled to the thioester group. In some embodiments the first member ofa specific binding pair is a protein or peptide having an N-terminalcysteine, and wherein the coupling step is carried out by reaction withthe N-terminal cysteine with the thioester by any suitable reaction,such as intein protein ligation (IPL) or native chemical ligation.

In some embodiments wherein the polymer is OEGMA, the OEGMA containscysteine groups, and the member of a specific binding pair contains aC-terminal thioester, the coupling step is carried out by reaction ofthe C-terminal cysteine with the thioester by any suitable reaction,such as intein protein ligation (IPL) or native chemical ligation.

In some embodiments of the invention, the surface portion comprises amaterial selected from the group consisting of metals, metal oxides,semiconductors, polymers, silicon, silicon oxide, and compositesthereof.

In some embodiments of the invention the linking layer is continuous; insome embodiments of the invention the linking layer is patterned. Insome embodiments of the invention the linking layer is a self-assembledmonolayer (SAM). In some embodiments of the invention the linking layercomprises an initiator-terminated alkanethiol.

In some embodiments of the invention the surface-initiatedpolymerization is carried out by atom transfer radical polymerization(ATRP); in some embodiments of the invention the surface-initiatedpolymerization is carried out by free radical polymerization.

In some embodiments, the article further comprises a protein, peptide,oligonucleotide or peptide nucleic acid covalently coupled to the brushmolecule. In some embodiments the protein, peptide, oligonucleotide orpeptide nucleic acid coupled to the brush molecule or to the surfaceconsist of or consist essentially of a single preselected molecule (thisis, one such molecule is coupled to the surface portion via the brushmolecule, to the exclusion of other different molecules). Thepreselected molecule may be a member of a specific binding pair, such asa receptor.

A further aspect of the invention is a method of detecting a secondmember of a specific binding pair nucleotide in a sample, comprising thesteps of: (a) providing a detector as described herein; (b) contacting asample (e.g., an aqueous sample or biological fluid) suspected ofcontaining the second member to the detector; and then (c) determiningthe presence or absence of binding of the second member to the firstmember, the presence of binding indicating the presence of the secondmember in the sample. The determining step can be carried out by anysuitable technique, but preferably involves directly or indirectlyconjugating an elongated nucleic acid to the second member, theelongated nucleic acid produced by the process of enzymatic extensionwith terminal transferase.

In some embodiments the first member is a probe nucleotide and secondmember is a target nucleotide. In such embodiments the determining stepmay comprise (i) elongating the target nucleotide with terminaltransferase to produce an elongated nucleic acid, and then (ii)detecting the presence or absence of the elongated nucleic acid.

In some embodiments the probe nucleotide comprises a beacon portion anda second portion, with the beacon portion folded by hybridization to thesecond portion, wherein the binding of the target nucleotide to theprobe nucleotide unfolds the beacon portion to produce a free endterminal for extension with terminal transferase. In such embodimentsthe deter mining step may comprise: (i) elongating the beacon portionwith terminal transferase to produce an elongated nucleic acid, and then(ii) detecting the presence or absence of the elongated nucleic acid.

In some embodiments the second member is a protein or peptide. In suchembodiments the determining step may comprise: (z) specifically bindingan antibody to the first member to form a sandwich, the antibody havinga first nucleic acid conjugated thereto, (ii) elongating the firstnucleici acid with terminal transferase to produce an elongated nucleicacid, and then (iii) detecting the presence or absence of the elongatednucleic acid.

An advantage of the foregoing methods is the variety of techniques bywhich the detecting step can be carried out. For example, the detectingstep may be carried out by: (a) ellipsometry; (b) surface plasmonresonance (SPR); (c) localized surface plasmon resonance using noblemetal nanoparticles in solution or on a transparent surface; (d) surfaceacoustic wave (SAW) devices; (e) quartz-crystal microbalance withdissipation (QCM-D) (e) atomic force microscopy, (f) detection ofradiolabeled nucleotides incorporated in the elongated portion forradioactive detection, (g) detection of stable isotope labelednucleotides in the elongated nucleic acid by mass spectrometry, (h)incorporation of fluorophores into the elongated nucleic acid andfluorescent detection, (z) incorporation of nucleotides containing aminoacids to which fluorophores are attached into the elongated nucleic acidand fluorescent detection, or (j) indirectly by hybridization of theelongated nucleic acid with a labeled complementary nucleic acid, andsubsequent detection of the complementary nucleic acid, wherein thecomplementary nucleic acid is labeled by coupling to a metalnanoparticle, quantum dot, fluorophore, or radionuclide, etc.

Still other aspects of the present invention are explained in greaterdetail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic illustration of “SI-ATRP of OEGMA” strategy forcreating functionalized non-fouling surfaces and molecular structure ofsilane initiator (11-(2-Bromo-2-methyl)propionyloxy)undecyltrichlorosilane.

FIG. 2. Schematic representation of microarray fabrication By NHS?EDCcoupling of antibodies to COOH-fucntionalized poly(OEGMA) brushes.

FIG. 3. Quasi-three dimensional, functionalized non-fouling films wereprepared via SI-ATRP of OEGMA from modified glass slides.

FIG. 4. The formation of initiator-silane SAM and poly(OEGMA) filmconfirmed by XPS.

FIG. 5. The protein resistance of the poly(OEGMA) brushes was tested bythe adsorption of fibronectin (Fn), bovine serum albumin (BSA), lysozyme(Ly) (all proteins at 1 mg/ml in PBS, pH=7.4), and undiluted fetalbovine serum (FBS).

FIG. 6 Surface initiated polymerization of OEGMA can be directly carriedout on a polymer substrate. (A) Chemical structures of Poly(OEGMA) andPVBC. Representative carbons are numbered to correspond with peaks in B.(B) XPS C(1s) spectra of PVBC coated substrates before (PVBC) and after(PVBC-POEGMA) SI-ATRP of OEGMA. The C(1s) spectrum of Poly(OEGMA) grownon silicon using a silane initiator (POEGMA) is shown for comparison.(C) Atomic percentages of carbon, oxygen and chlorine found in eachsample.

FIG. 7. Alternative embodiment of a protein microarray by printing ofantibodies or other receptors to the polymer brush and their covalentattachment to activated OH groups of poly(OEGMA) brush. (A) Reactionscheme of coupling protein to CDI-activated OEGMA on glass. (B) Printingof an IgG capture antibody on brush and effect of washing of surface onspot stability. (C) Comparison of IL4 detection on poly(OEGMA) brush andon a FAST™ slide (Whatman).

FIG. 8. Applications of TdTase-mediated DNA-extension signalamplification method.

FIG. 9. Surface initiated extension of DNA by terminal transferase onbiomolecular nanoarrays generated by e-beam lithography.

FIG. 10. Tapping mode AFM images in air for biomolecular arrays ofdifferent feature sizes (A-B: 0.1 μm; C-D: 4 μm) with 5′-SH—(CH₂)₆-T25SAM after a 2-hour incubation with active terminal transferase (TdTase).

FIG. 11. SPR sensorgram for TdTase-mediated signal amplification with anantibody covalently conjugated with oligonucleotides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

The disclosures of all United States patents cited herein areincorporated by reference in their entirety.

1. Definitions

“SI-ATRP” as used herein means surface initiated atom transfer radicalpolymerization.

“OEGMA” as used herein refers to oligo(ethylene glycol)methylmethacrylate.

“Biological fluid” as used herein may be any fluid of human or animalorigin, including but not limited to blood, blood plasma, peritonealfluid, cerebrospinal fluid, tear, mucus, and lymph fluid. Biologicalfluids generally contain a mixture of different proteins therein, andtypically contain other constituents such as other cells and molecules.Biological fluids may be in their natural state or in a modified stateby the addition of ingredients such as reagents or removal of one ormore natural constituents (e.g., blood plasma).

“Kosmotrope”, while originally used to denote a solute that stabilized aprotein or membrane, is also used by those skilled in the art, and isused herein, to denote a substituent or “head group” which, whendeposited on a surface, renders that surface protein-resistant. See,e.g., R. Kane. P. Deschatelets and G. Whitesides, Kosmotropes Form theBasis of Protein-Resistant Surfaces, Langmuir 19, 2388-2391 (2003).Numerous kosmotropes are known and examples include but are not limitedto OEGMA.

“Polymer” as used herein is intended to encompass any type of polymer,including homopolymers, heteropolymers, co-polymers, ter-polymers, etc.,and blends, combinations and mixtures thereof.

“Specific binding pair” as used herein refers to two compounds thatspecifically bind to one another, such as (functionally): a receptor anda ligand (such as a drug), an antibody and an antigen, etc.; or(structurally): protein or peptide and protein or peptide; protein orpeptide and nucleic acid; and nucleotide and nucleotide etc.

2. Substrates

The present invention can be utilized to forms surfaces on a variety ofdifferent types of substrates.

In some embodiments, the article is a label-free optical or massdetector (e.g., a surface plasmon resonance energy detector, an opticalwave guide, an ellipsometry detector, etc.) and the surface is a sensingsurface (e.g., a surface portion that would be in contact with abiological fluid). Examples of such articles include but are not limitedto those described in U.S. Pat. Nos. 6,579,721; 6,573,107; 6,570,657;6,423,055; 5,991,048; 5,822,073; 5,815,278; 5,625,455; 5,485,277;5,415,842; 4,844,613; and 4,822,135.

In other embodiments, the article is a biosensor, an assay plate, or thelike. For example, the present invention may be utilized with opticalbiosensors such as described in U.S. Pat. Nos. 5,313,264 to Ulf et al.,5,846,842 to Herron et al., 5,496,701 to Pollard-Knight et al., etc. Thepresent invention may be utilized with potentiometric or electrochemicalbiosensors, such as described in U.S. Pat. No. 5,413,690 to Kost, or PCTApplication WO98/35232 to Fowlkes and Thorp. The present invention maybe utilized with a diamond film biosensor, such as described in U.S.Pat. No. 5,777,372 Thus, the solid support may be organic or inorganic;may be metal (e.g., copper or silver) or non-metal; may be a polymer ornonpolymer; may be conducting, semiconducting or nonconducting(insulating); may be reflecting or nonreflecting; may be porous ornonporous; etc. For example, the solid support may be comprised ofpolyethylene, polytetrafluoroethylene, polystyrene, polyethyleneterephthalate, polycarbonate, gold, silicon, silicon oxide, siliconoxynitride, indium, tantalum oxide, niobium oxide, titanium, titaniumoxide, platinum, iridium, indium tin oxide, diamond or diamond-likefilm, etc.

The present invention may be utilized with substrates for “chip-based”and “pin-based” combinatorial chemistry techniques. All can be preparedin accordance with known techniques. See. e.g., U.S. Pat. No. 5,445,934to Fodor et al., U.S. Pat. No. 5,288,514 to Ellman, and U.S. Pat. No.5,624,711 to Sundberg et al., the disclosures of which are incorporatedby reference herein in their entirety.

Substrates as described above can be formed of any suitable material,including but not limited to a material selected from the groupconsisting of metals, metal oxides, semiconductors, polymers(particularly organic polymers in any suitable form including woven,nonwoven, molded, extruded, cast, etc.), silicon, silicon oxide, andcomposites thereof.

Polymers used to form substrates as described herein may be any suitablepolymer, including but not limited to: poly(ethylene) (PE),poly(propylene) (PP), cis and trans isomers of poly(butadiene) (PB), cisand trans isomers of poly(ispoprene), poly(ethylene terephthalate)(PET), polystyrene (PS), polycarbonate (PC), poly(epsilon-caprolactone)(PECL or PCL), poly(methyl methacrylate) (PMMA) and its homologs,poly(methyl acrylate) and its homologs, poly(lactic acid) (PLA),poly(glycolic acid), polyorthoesters, poly(anhydrides), nylon,polyimides, polydimethylsiloxane (PDMS), polybutadiene (PB),polyvinylalcohol (PVA), polyacrylamide and its homologs such aspoly(N-isopropyl acrylamide), fluorinated polyacrylate (PFOA),poly(ethylene-butylene) (PEB), poly(styrene-acrylonitrile) (SAN),polytetrafluoroethylene (PTFE) and its derivatives, polyolefinplastomers, and combinations and copolymers thereof, etc.

If desired or necessary, the substrate may have an additional layer suchas a gold or an oxide layer formed on the relevant surface portion tofacilitate the deposition of the linking layer, as discussed furtherbelow.

3. Linking (or “Anchor”) Layers

Anchor layers used to carry out the present invention are generallyfouled from a compound comprising an anchor group coupled (e.g.,covalently coupled) to an initiator (e.g., directly coupled or coupledthrough an intermediate linking group). The choice of anchor group willdepend upon the surface portion on which the linking layer is formed,and the choice of initiator will depend upon the particular reactionused to faun the brush polymer as discussed in greater detail below.

The anchoring group may be selected to covalently or non-covalentlycouple the compound or linking layer to the surface portion.Non-covalent coupling may be by any suitable secondary interaction,including but not limited to hydrophobic bonding, hydrogen bonding, Vander Waals interactions, ionic bonding, etc.

Examples of substrate materials and corresponding anchoring groupsinclude, for example, gold, silver, copper, cadmium, zinc, palladium,platinum, mercury, lead, iron, chromium, manganese, tungsten, and anyalloys thereof with sulfur-containing functional groups such as thiols,sulfides, disulfides (e.g., —SR or —SSR where R is H of alkyl, typicallylower alkyl, or aryl), and the like; doped or undoped silicon withsilanes and chlorosilanes (e.g., —SiR₂Cl wherein R is H or alkyl,typically lower alkyl, or aryl); metal oxides such as silica, alumina,quartz, glass, and the like with carboxylic acids as anchoring groups;platinum and palladium with nitrites and isonitriles; and copper withhydroxamic acids. Additional suitable functional groups suitable as theanchoring group include benzophenones, acid chlorides, anhydrides,epoxides, sulfonyl groups, phosphoryl groups, hydroxyl groups,phosphonates, phosphonic acids, amino acid groups, amides, and the like.See, e.g., U.S. Pat. No. 6,413,587.

Any suitable initiator may be incorporated into the anchoring group byintroduction of a covalent bond at a location non-critical for theactivity of the initiator. Examples of such initiators include, but arenot limited to, bromoisobutyrate, polymethyl methacrylate-Cl,polystyrene-Cl, AIBN, 2-bromoisobutyrate, chlorobenzene, hexabromomethylbenzene, hexachloromethyl benzene, dibromoxylene, methylbromoproprionate. Additional examples of initiators include thoseinitiators described in U.S. Pat. No. 6,413,587 to Hawker (particularlyat columns 10-11 thereof) and those initiators described in U.S. Pat.No. 6,541,580 to Matyjaszewski et al.

As noted above, a linking group or “spacer” may be inserted between theanchoring group and initiator. The linker may be polar, nonpolar,positively charged, negatively charged or uncharged, and may be, forexample, saturated or unsaturated, linear or branched alkylene,aralkylene, alkarylene, or other hydrocarbylene, such as halogenatedhydrocarbylene, particularly fluorinated hydrocarbylene. Preferredlinkers are simply saturated alkylene of 3 to 20 carbon atoms, i.e.,—(CH₂)₄— where n is an integer of 3 to 20 inclusive. See, e.g., U.S.Pat. No. 6,413,587. Another preferred embodiment of the linker is anoligoethyleneglycol of 3 to 20 units, i.e., (CH₂CH₂O)_(n) where n rangesfrom 3 to 20.

The anchoring layer may be deposited by any suitable technique. It maybe deposited as a self-assembled monolayer. It may be created bymodification of the substrate by chemical reaction (see, e.g., U.S. Pat.No. 6,444,254 to Chilkoti et al.) or by reactive plasma etching orcorona discharge treatment. It may be deposited by a plasma depositionprocess. It may be deposited by spin coating or dip coating. It may bedeposited by spray painting. It may also be deposited by deposition,printing, stamping, etc. It may be deposited as a continuous layer or asa discontinuous (e.g., patterned) layer.

In some preferred embodiments, the substrate is glass, silicon oxide orother inorganic or semiconductor material (e.g., silicon oxide, siliconnitride) and compound semiconductors (e.g., gallium arsenide, and indiumgallium arsenide) used for microarray production.

In some preferred embodiment, the anchoring group is a silane orchlorosilane (e.g., —SiR₂Cl wherein R is H or alkyl, typically loweralkyl, or aryl).

4. Brush Polymer Formation

The brush polymers are, in general, formed by the polymerization ofmonomeric core groups having a protein-resistant head group coupledthereto.

Any suitable core vinyl monomer polymerizable by the processes discussedbelow can be used, including but not limited to styrenes,acrylonitriles, acetates, acrylates, methacrylates, acrylamides,methacrylamides, vinyl alcohols, vinyl acids, and combinations thereof.

Protein resistant groups may be hydrophilic head groups or kosmotropes.Examples include but are not limited to oligosaccharides, tri(propylsulfoxide), hydroxyl, glycerol, phosphorylcholine, tri(sarcosine)(Sarc), N-acetylpiperazine, permethylated sorbitol,hexamethylphosphoramide, an intramolecular zwitterion (for example,—CH₂N⁺(CH₃)₂CH₂CH₂CH₂SO₃ ⁻) (ZW), and mannitol.

Additional examples of kosmotrope protein resistant head groups include,but are not limited to:

-(EG)₆OH;

—O(Mannitol);

—C(O)N(CH₃)CH₂(CH(OCH₃))₄CH₂OCH₃;

—N(CH₃)₃ ⁺Cl⁻/—SO₃ ⁻Na⁺;

—N(CH₃)₂ ⁺CH₂CH₂SO₃;

—C(O)Pip(NAc);

—N(CH₃)₂ ⁺CH₂CO₂;

—O([Blc-α(1,4)-Glc-β(1)-]);

—C(O)(N(CH₃)CH₂C(O))₃N(CH₃)₂;

—N(CH₃)₂ ⁺CH₂CH₂CH₂SO₃ ⁻;

—C(O)N(CH₃)CH₂CH2N(CH₃)P(O)(N(CH₃)₂)₂; and

—(S(O)CH₂CH₂CH₂)₃S(O)CH₃.

See, e.g., R. Kane et al., Langmuir 19, 2388-91 (2003)(Table 1).

A particularly preferred protein resistant head group is poly(ethyleneglycol), or “PEG”, for example PEG consisting of from 3 to 20 monomericunits.

Free radical polymerization of monomers to form brush polymers can becarried out in accordance with known techniques, such as described inU.S. Pat. No. 6,423,465 to Hawker et al.; U.S. Pat. No. 6,413,587 toHawker et al.; U.S. Pat. No. 6,649,138 to Adams et al.; US PatentApplication 2003/0108879 to Klaerner et al.; or variations thereof whichwill be apparent to skilled persons based on the disclosure providedherein.

Atom or transfer radical polymerization of monomers to fowl brushpolymers can be carried out in accordance with known techniques, such asdescribed in U.S. Pat. No. 6,541,580 to Matyjaszewski et al.; U.S. Pat.No. 6,512,060 to Matyjaszewski et al.; or US Patent Application2003/0185741 to Matyjaszewski et al., or variations thereof which willbe apparent to skilled persons based on the disclosure provided herein.

In general, the brush molecules formed by the processes described hereinwill be from 2 or 5 up to 50 or 100 nanometers in length, or more, andwill be deposited on the surface portion at a density of from 10, 20 or40 up to 100, 200 or 500 milligrams per meter², or more.

In some preferred embodiments, the polymer layer is formed by SI-ATRP ofOEGMA to form a poly(OEGMA) film. In particularly preferred embodiments,the polymer layer is a functionalized poly(OEGMA) film prepared(preferably in a single step) by copolymerization of a methacrylate andmethyl terminated OEGMA. For the copolymer, poly(sodiummethacrylate-co-OEGMA), or the like, the carboxylate can be converted tocarboxyl acid by incubation of the copolymer in an acid such as HCl,resulting in poly(MAA-co-OEGMA)(MAA: methacrylate acid). Thepoly(MAA-co-OEGMA) can be further converted to the ester in accordancewith known techniques.

In other particularly preferred embodiment, copolymers ofmethoxy-terminated OEGMA with OH-terminated OEGMA, and the OH isdirectly used to conjugate molecules of all types via their availableamine groups using well known coupling reactions, such as tresylchloride conjugation and CDI chemistry. In other particularly preferredembodiments, copolymers of methoxy-OEGMA and OH-terminated OEGMA aresynthesized on the substrate by SI-ATRP, and the OH groups are convertedto COOH groups by reaction with N-hydroxy succinimide (NHS), and theCOOH groups are used as sites for attachment of molecules via availableamine groups on the molecule.

In other preferred embodiments, the OEGMA is synthesized to directlycontain a fraction of thioester groups, so as to permit directattachment of any molecule but preferentially a protein or peptide viareaction with their N-terminal cysteine using intein protein ligation(IPL) or native chemical ligation. Similarly, this approach can bereversed, so that the OEGMA polymer could contain, either as aconsequence of incorporation during SI-ATRP using a suitableOEGMA-functionalized monomer or by conversion of incorporated groups(e.g., but not limited to OH or COOH groups) to a cysteine, so that amolecules such as a protein or peptide contains a C-terminal thioester,as is typically obtained by intein-mediated cleavage of a target proteinor peptide of an intein fusion could be covalently attached via itsthioester to the cysteine groups presented on the termini of the OEGMApolymer.

Proteins, peptides, antibodies, oligonucleotides or nucleic acids (suchas DNAs) (e.g., 3-50 nucleotides in length) or other members of abinding pair can be deposited on the polymer layer, typically afterintroduction of a carboxyl group therein, by any suitable technique suchas microprinting or microstamping. Microarrays or nanoarrays ofoligonucleotides can be formed on the substrates by any suitabletechnique, such as e-beam lithography.

5. Uses and Applications of Articles

In some embodiments the present invention is utilized by (a) providingan article as described herein, the article further comprising a firstmember of a specific binding pair such as a protein, peptide,oligonucleotide, peptide nucleic acid or the like covalently coupled tothe brush molecule, the first member preferably consisting essentiallyof a single preselected molecule; and then (b) contacting the article toa biological fluid or other composition containing a second member ofthe specific binding pair, wherein the second member of the specificbinding pair binds to the surface portions. Such uses are particularlyappropriate where the article is a sensor or biosensor as described ingreater detail above.

Binding of the second member of the specific binding pair can beachieved by any suitable technique. In some embodiments the binding ispreferably carried out by sandwich assay. In some embodiments thebinding may be detected by extension of a nucleic acid such as a DNAwith terminal transferase to form an extended nucleic acid, whichextended nucleic acid may be detected by any suitable technique.Terminal transferase (e.g., TdTase) is known, and methods of elongatingnucleic acids (or shorter oligonucleotides), to produce extended nucleicacids (or longer oligonucleotides) or extension products, and methods ofdetecting such extension products can be carried out in accordance withknown techniques or variations thereof that will be apparent to thoseskilled in the art in view of the instant disclosure and known methodsof using terminal transferase as described in (for example) U.S. Pat.Nos. 6,911,305; 6,864,060; 6,709,816; 6,642,375; 6,406,890; 6,323,337;6,136,531; 5,824,514; 5,397,698; and 5,344,757.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXPERIMENTAL

The science community has recently witnessed an explosive development ofmicroarray technique as one of the high-throughput screening strategies.Although with great success, the development of microarray technique isalso accompanied with obstacles. One particular bottleneck for thedevelopment of microarray technique is the surface chemistry ofmicroarray substrate. Typical materials used as microarray substrates,for example, poly(vinylidene fluoride), nitrocellulose, nylon,poly(L-lysine), silane, and ethylene-glycol self-assembled monolayersuffer from problems such as high level of nonspecific adsorption and/orlow loading density (Zhu, Jun et al. 2001). Here, we have successfullydemonstrated the “modular design of initiator” strategy to be generallyapplicable in creating non-fouling surfaces using poly(MAA-co-OEGMA)(FIG. 1). To highlight the potential applications of this non-foulingsurface, we incorporated functional groups into the coating materials asbinding sites and conjugated them with antibodies for the detection ofcytokines in a microarray format (FIG. 2). Other detection schemes usingDNA/RNA aptamers and enzymatic amplification can be easily incorporatedinto this surface modification platform to further improve the detectionsensitivity (FIG. 8). Other than proteins, this technology is alsoapplicable to micro- and nano-array detection systems for otherbiomolecules such as cDNA, (Schena, Shalon et al. 1995) small molecules,(MacBeath, Koehler et al. 1999) peptides, (Houseman, Huh et al. 2002)oligosaccharides, (Fukui, Feizi et al. 2002) and carbohydrates,(Houseman and Mrksich 2002; Wang, Liu et al. 2002), which have beendemonstrated to be powerful tools in drug discovery, medicaldiagnostics, proteomic and genomic profiling, and other biotechnologicalapplications.

FIG. 1. Schematic illustration of “SI-ATRP of OEGMA” strategy forcreating functionalized non-fouling surfaces and molecular structure ofsilane initiator (11-(2-Bromo-2-methyl)propionyloxy)undecyltrichlorosilane. SiO₂ wafers were cleaned with a mixture ofNH₄OH:H₂O₂:H₂O (1:1:5, v/v/v) (Ma, Hyun et al. 2004) and glass slideswere cleaned with al % RBS solution and a 1:1 (v/v) mixture ofmethanol:HCl before used immediately for silanization. Under N₂atmosphere, the initiator-silane was deposited on silicon oxidesubstrate by vapor deposition and on cleaned glass slides by incubatingwith silane in anhydrous tetrahydrofuran. SI-ATRP was carried out asreported previously (Perruchot, Khan et al. 2001; Ma, Hyun et al. 2004).Briefly, under nitrogen, polymerization was initiated by adding thereaction mixture containing CuBr, bipyridine, and OEGMA in deionizedwater and methanol to the flask and was continued for a specified time.The samples were pulled out of the solution to stop the polymerization,rinsed with methanol and dried under flowing N₂ gas.

FIG. 2. Schematic representation of microarray fabrication. The carboxylfunctionalized polymer coated slides were immersed into HCl solution andthen NHS/EDAC solution. Each test slide was robotically printed with 12identical 8×5 arrays using a Microsys 5100 microarrayer in anatmospherically controlled chamber. For each array, rows 1 and 8 werebiotinylated BSA (B-BSA), regarded as positive control. Row 2 was BSA asnegative control. Rows 3-7 were capture antibodies for human IL-1β,TNF-α, VEGF, IL-6, and TGF-(31, respectively. Prior to printing, the pinwas sequentially cleaned in 5% Micro-Cleaning Solution. The printedarrays were immersed into sodium bicarbonate buffer (pH=8.5) fordeactivation. Stock solutions of cocktail of cytokines/growth factorswere prepared and diluted for dose-response studies. Each slide wasdirectly incubated with cytokine solution. After three aspiration/washcycles, each array was incubated with a cocktail containing a mixture offive biotinylated detection antibodies and then with streptavidin-Cy5.Finally, slides were scanned, imaged, and analyzed using a GenePix 4000Bmicroarray scanner and GenePix 5.0 software.

FIG. 3. Quasi-three dimensional, functionalized non-fouling films wereprepared via SI-ATRP of OEGMA from modified glass slides. Functionalizedpoly(OEGMA) films were prepared in one step by copolymerization ofsodium methacrylate and methyl terminated OEGMA. In order to achieve ahigher density, the silane initiator layer on glass was formed bydipping glass slides into silane initiator solution instead of vapordeposition. For copolymer poly(sodium methacrylate-co-OEGMA), thecarboxylate was converted to carboxyl acid by incubation of copolymer in1 M HCl, resulted in poly(MAA-co-OEGMA) (MAA: methyacrylate acid). Thispoly(MAA-co-OEGMA) was further converted to ester by incubation inN-hydroxysuccinimide/N-ethyl-N'-(3-dimethylaminopropyl) carbodiimidehydrochloride (NHS/EDC) solution.

FIG. 4. The formation of initiator-silane SAM and poly(OEGMA) film wasconfirmed by XPS (A). The polymerization introduced unique Br peaks(with an atomic percentage (AT. %) of ˜5 AT. % based on Br_(3d) at ˜69eV) at the low end of binding energy (BE) in the survey scan ofinitiator-silane SAM when compared with the spectrum of silicon oxidesurface, and also increased carbon content from 1.2 to 49.3 AT. %. Thelater caused the AT. % of Si_(2s) to drop from 18.3 for silicon oxidesurface to 17.8 for a thin film and further down to 4.3 for a thick filmdue to enhanced attenuation effect posed by the increased poly(OEGMA)thickness. The poly(OEGMA) films (thickness of 95 Å) exhibited an XPSC/O ratio of 2.5, which is somewhat greater than the stoichiometricvalue of 2.1 for poly(OEGMA).

The polymerization and conversion of poly(sodium methacrylate-co-OEGMA)(thickness ˜10 nm) was also confirmed by XPS study (B). Before and afterNHS/EDC activation, survey scan showed no difference for poly(OEGMA)while the same treatment showed a new N₁, peak at ˜399 eV forfunctionalized copolymer coating. The final ratio of OEGMA and sodiummethacrylate is determined using XPS core scan: one can roughly estimatethe final composition to be 1/1.5 for OEGMA/sodium methacrylate, whichis dramatically different from the initial feed ratio of 50/1. Thisradical change is probably due to the difference in polymerizationkinetics for the two different species: OEGMA has a low incorporationrate because of its steric hindrance. The conversion of COOH to NHS isassumed to be 100%.

FIG. 5. The protein resistance of the poly(OEGMA) brushes was tested bythe adsorption of fibronectin (Fn), bovine serum albumin (BSA), lysozyme(Ly) (all proteins at 1 mg/ml in PBS, pH=7.4), and undiluted fetalbovine serum (FBS). Poly(OEGMA) modified and the control(initiator-modified) silicon wafers were immersed into a proteinsolution for 1 h, rinsed with buffer, dried under nitrogen stream, andthe thickness of the adsorbed protein layer was measured byellipsometry. The thickness of the protein layer (d_(y)) is a functionof the poly(OEGMA) film thickness (d_(x)). The thickness of the adsorbedprotein on the control surface varied depending upon the protein,ranging from ˜10 Å (Ly) to ˜25 Å for the other proteins and serum. Incontrast, the poly(OEGMA) brush with ˜14 Å thickness showedsignificantly less protein adsorption of all proteins and of serum.Increasing the thickness of the poly(OEGMA) brush to ˜95 Å (and beyond,data not shown) eliminated the adsorption of all proteins and mostnotably that of serum to below the 1 Å detection limit of ellipsometry.These results are consistent with our previous results (Ma, Hyun et al.2004) that a dense and thick poly(OEGMA) brush completely resistsprotein adsorption from solution.

FIG. 6. OEGMA brushes were polymerized directly on a spun-cast film ofpoly(vinylbenzyl chloride) (PVBC) film without initiator to demonstratethat SI-ATRP can be performed on polymers in a straightforward andfacile manner. Briefly, clean glass slides were treated with adhesionpromoter hexamethyldisilazane followed by spinning on a 1% (w/v)solution of PVBC in toluene at 3000 rpm for one minute, and then dryingat 80° C. for 20 min Ellipsometric measurements showed uniform PVBC filmthicknesses of 35=1 nm. The OEGMA polymerization was carried out in anoxygen-free environment using CuBr/bipyridine as catalyst in awater/methanol mixture containing OEGMA monomer. Figure C.2.8B shows theXPS C(1s) spectra of PVBC coated substrates before (PVBC) and after(PVBC-POEGMA) SI-ATRP of OEGMA. The C(1s) spectrum of Poly(OEGMA)brushes grown on silicon using a silane initiator (POEGMA) is shown forcomparison. The PVBC-POEGMA spectrum exhibits the formation of an esterpeak at 289 eV, as well as a shift in the peaks at 286.7 and 285 eV, toclosely mirror the spectrum of Poly(OEGMA) grown from silane initiator.Furthermore, the close match in carbon and oxygen atomic percentagesfound in the PVBC-POEGMA (70.5% C/29.0% O) and POEGMA (70.0% C/29.9% O)samples contrasted sharply with the atomic percentages found in the PVBCsample (91.5% C/0.5% O). The results indicate that halogenated polymers,be they intrinsic or covalently grafted, can be modified with a coatingof poly(OEGMA) through the use of dip or spin coated halogenated films.

FIG. 7. SI-ATRP of hydroxyl terminated OEGMA on glass and siliconyielded OEGMA films with an ellipsometric thickness of 10 nm. Thehydroxyl groups of the polymer brush were then activated with a 0.5Msolution of 1,1-Carbonyldiimidazole (CDI) in dry dioxane at 37° C. for 2hours. The conversion of hydroxyl terminated OEGMA to OEGMA-CDI wasconfirmed by the appearance of a new N1s XPS peak at ˜399 eV due to thepresence of the imidazolyl carbamate group introduced during CDIactivation. This intermediate imidazolyl carbamate is readily displacedby amine-containing proteins, resulting in stable carbamate linkagesbetween OEGMA coatings and proteins. Attachment of proteins to thenonfouling OEGMA coatings on glass was achieved by introducingchemically reactive carbonyldiimidazole (CDI) groups into thepolymerized brush. SI-ATRP of OEGMA yielded films of hydroxy-terminatedethylene glycol “stalks” on a methacrylate “core” (see FIG. 7A).Proteins were then conjugated through accessible amino groups FIG. 7Bshows the fluorescence image of Cy-5 labeled anti-IL-4 capture antibodyrobotically printed and chemically tethered to CDI-activated OEGMAbrushes on glass before and after a wash step. FIG. 7C compares thedose-response curves of IL-4 fluoroimmunoassay on CDT-activated OEGMAmicroarray and on a commercially available nitrocellulose microarrayslide (FAST, Whatman). Features to note here are (1) the regionsadjacent to the printed capture antibody in FIG. 6B are virtually blackowing to the ultra-low background fluorescence of poly(OEGMA), (2) thepoly(OEGMA) microarray is greater than 10-100× more sensitive than FASTslides owing to the complete resistance of the poly(OEGMA) to thenon-specific adsorption of protein during the analyte incubationprocess, and (3) CDI immobilization chemistry clearly retains theactivity of the tethered capture antibody (poly(OEGMA) microarrays candetect down to 1 pg/ml IL-4!).

FIG. 8. Applications of TdTase-mediated DNA-extension signalamplification method. The antibody-based sandwich-type protein detectionscheme (A) can be easily combined or replaced with our technique. Forexample, the secondary antibody can be attached with shortoligonucleotides (B) or substituted with an aptamer designed for atarget molecule (C), and then extended at the 3′ end by TdTase. Fornucleic acid detection, target genes can be chemically grafted at their3′ end to a surface and cDNA that is hybridized to the target gene canbe extended at its 3′ end for signal amplification (D). In addition,molecular beacons containing target genes can be chemically grafted to asurface at their 5′ end. The binding of cDNA to the target gene causesunfolding of the molecular beacon so that its 3′ end becomes accessiblefor extension by TdTase (E).

FIG. 9. Surface initiated extension of DNA by terminal transferase onbiomolecular nanoarrays generated by e-beam lithography. Anelectron-sensitive resist layer (130 nm) of poly(methylmethacrylate) wasspin-coated onto the cleaned Si substrate and annealed, and thenpatterned by exposure to an electron beam using a scanning electronmicroscope (A). A layer of chromium (50 Å) and a layer of gold (300 Å)were deposited by e-beam evaporation onto the patterned PMMA/Si toobtain geometrically well-defined gold features on the exposed SiO₂surface (B). The gold-coated resist was lifted-off, leaving behind goldpatterns of 35 nm height on the Si substrate (C). A self-assembledmonolayer (SAM) of an oligonucleotide was prepared on the patterned goldarrays by incubation in a reduced DNA-thiol solution overnight (D).Enzymatic DNA extension was performed by incubating the patternedoligonucleotide arrays with TdTase in the presence of cobalt-containingbuffer and 2′-deoxyguanosine 5′-triphosphate (dTTP) at 37° C. for 2 h(E).

FIG. 10. Tapping mode AFM images in air for biomolecular arrays ofdifferent feature sizes (A-B: 0.1 μm; C-D: 4 μm) with 5″-SH—(CH2)6-T25SAM after a 2-hour incubation with active terminal transferase (TdTase).Insets are the images of A and C at a higher magnification (1 μm×1 μm).B and D are the line profiles of A and C. Dotted lines represent theaverage height of gold arrays and immobilized DNA SAM. Poly(T)oligonucleotides were successfully extended on gold arrays withdifferent lateral feature sizes. There are four significant observationsfrom these experiments: (1) The average heights of the synthesized DNAnanostructures on gold features of 0.1 and 4 μm sides (minus the averageheight of gold arrays and immobilized DNA SAM) were 45.5±5.2 nm and120.7±9.3 nm, respectively (B and D). The significant height increasesuggests that the TdTase catalyzed polymerization of dTTP wassuccessful. (2) The height of the DNA nanostructures grown by TdTaseappears to be dependent upon the lateral feature size of the underlyinggold patterns, with significantly lower extension observed for smallerfeature size. This trend might be related to decreased stericaccessibility of TdTase for the immobilized DNA as the feature sizebecomes smaller. (3) Images at a higher magnification for the 100 nmgold features showed that the DNA nanostructures had a lateral featuresize of 358.5±10.0 nm (full width at half maximum, Figure A inset) andexhibited significant lateral extension. (4) DNA growth appeared to beheterogeneous and resulted in a layer of DNA with a RMS roughness of13.6±0.6 nm for the 4 μm gold features (Figure C inset) vs. 0.23±0.02 nmfor bare gold. In addition, these DNA nanostructures were less compactthan those for 100 nm gold features, suggesting that their molecularweights were also affected by the lateral feature size.

FIG. 11. To test whether TdTase can extend oligonucleotides conjugatedto an antibody, we ran a SPR experiment with a DNA-antibody complexconjugated through streptavidin-biotin interactions. Twenty microlitersof biotinylated goat α-mouse antibody at 100 μg/ml was injected to abare gold SPR chip (5 nm Cr and 35 nm Au), followed by a 10-minute PBSwash. Binding measurements and extension reactions were run at 37° C. atflowrate of 1 μl/min with PBS as washing buffer. Twenty microliters ofstreptavidin at 100 μg/ml, twenty microliters of biotin-T₂₅ at 10 μM,and sixty microliters of TdTase (0.6 U/pd) and dTTP (1 mM) incobalt-containing buffer were injected sequentially, with a 10-minutePBS wash after each injection. There was an increase in RU of ˜4,000,indicating that DNA conjugated on the antibody can be extended by TdTase(FIG. 3A).

In some embodiments, the present invention provides one or more of thefollowing features:

1) Fast and stable immobilization. For most surface substrates, capturereagents are usually physisorbed on the surface. In contrast,poly(MAA-co-OEGMA) films provide a convenient way of covalentlyimmobilizing capture reagents on the surface without protection andde-protection chemistry. The ester group (C(O)—NHS) is well known forfast protein coupling (Hermanson 1996) and can be easily introduced tothe poly(MAA-co-OEGMA) platform through a two-step chemistry (FIG. 2).The superior tolerance of surface-initiated atom transfer radicalpolymerization (SI-ATRP) to functional groups exhibited in this case asthe direct introduction of COOH terminal functional group. The reactionhas been demonstrated to complete within a few seconds under amoisturized environment instead of aqueous surrounding (Hyun, Ahn et al.2002). The excess C(O)—NHS groups can be easily deactivated byincubation of sodium bicarbonate buffer (pH=8.5), which is better thanaldehyde slides that used BSA to neutralize (MacBeath and Schreiber2000). To further optimize the polymer matrix, methyl terminated OEGMA(n=9) and OH terminated OEGMA (n=12) were copolymerized. The different nvalues were chosen so that OH functional will be more accessible for theimmobilization of protein and other ligands. The OH functional group caneither be directly used or be further converted to COOH group asdemonstrated in our previous paper (Hyun, Ma et al. 2002). In analternative approach, proteins can be coupled through their amine groupsto the OH groups present in the poly(OEGMA) after activation of the OHgroups using CDI (Hermanson 1996) (FIG. 7A). This approach givesconsistent and reproducible attachment of proteins.

2) Low and consistent background. Poly(MAA-co-OEGMA), unlikenitrocellulose-coated, aldehyde-coated, epoxy-coated and silylatedslides (Li, Nath et al. 2003; Li and Reichert 2003), has minimal levelof auto-fluorescence emission, of which majority is originated from theunderlying glass substrate. In addition, poly(MAA-co-OEGMA) films showedno sign of nonspecific adsorption of detection reagents (antibodies) tosubstrates (FIGS. 5 and 6). The same is true of the CDI-activatedprotein microarray, as seen in FIG. 7C. These films also showed aconsistent level of background, regardless of cytokine concentration (10pg/ml to 10 ng/ml), even for cytokines in serum (˜55 mg/ml proteinmixture) (Elbert and Hubbell 1996), or E. coli lysate, and is very closeto their unprocessed counterpart, even without any blocking step. Theoverall background of poly(MAA-co-OEGMA)-coated surfaces and othersimilar functionalized poly(OEGMA)-based matrixes is 100 times lowerthan that of nitrocellulose-coated one (FAST™ slides). The advantages oflow and consistent background are: 1) it enables the detection of atarget biomolecule in complex biological fluids such as whole blood,serum, or cell lysate and 2) low auto fluorescence also improvesdetection sensitivity and increases the detection limit for variousassays. Current detection limits of better than 0.1 pg/ml cytokine canbe easily achieved.

3) High loading density. The loading density of capture reagent, targetbiomolecules, and detection reagent is substantially increased as aresult of the increased surface to volume ratio for the quasi threedimensional poly(MAA-co-OEGMA) films (FIG. 2) when compared with planarEG-SAM based substrates (Houseman and Mrksich 2002), but lower than the3D gel pad system, which unfortunately suffers from the problem of highlevel of nonspecific adsorption (Arenkov, Kukhtin et al. 2000). OEGterminated SAM (Houseman and Mrksich 2002) as well as PLL-g-PEG(Ruiz-Taylor, Martin et al. 2001) system have been used but showedlimited success. The former suffered from low loading density and thelater suffered from poor performance under complex solution (FBS) due toits low density of PEG coating as well as limited stability.

4) Applicable for biomolecular detection in the microarray and nanoarrayformat. The ultra-low background of poly(MAA-co-OEGMA) films not onlyeliminates the need of blocking, but also significantly improves thequality of the signal originated from the target biomolecules (FIGS. 2and 7). Bovine serum albumin (BSA) is widely used as a blocking materialbut there is evidence shown that adsorbed BSA affects protein binding orligand-receptor interaction (MacBeath, Koehler et al. 1999). Theelimination of the blocking step provides substantial benefit forbiomolecular detection, especially for microarrays with spot size ofsub-micron length scale, where the signal-to-noise ratios can beseverely reduced by the presence of BSA molecules.

5) Application to other receptor-analyte pairs. The sandwich-typedetection scheme employed on our poly(MAA-co-OEGMA) platform is notlimited to the use of antibodies as capture and detection agents. Otherdetection modalities such as DNA/RNA aptamers can be easily incorporatedand further modified to the detection of other biomolecules. Theadvantages of using DNA/RNA aptamers are their superior thermal andchemical stability, lower cost of synthesis, and ease of modification toachieve high binding affinity (Bock, Griffin et al. 1992; Macaya,Schultze et al. 1993).

6) Signal amplification for antibody-based biomolecular detection. Toachieve ultra-high sensitivity, we developed an amplification techniquewith terminal deoxynucleotidyl transferase (TdTase), an enzyme thatrepeatedly adds mononucleotides to the 3′ end of single- ordouble-stranded DNA/RNA (FIG. 8). The addition of these nucleotides attheir 3′ end of the target DNA significantly increases the detectablesignal. There are several ways that we can combine or replaceantibody-based sandwich-type protein detection methods with signalamplification by TdTase-mediated DNA extension: 1) secondary antibodiescan be attached with short oligonucleotides, which are amplified byTdTase. Short oligonucleotides can be randomly attached to lysineresides of antibodies or site-specifically attached to the Fc regiononly, so that the extension of multiple oligonucleotides on the antibodywould provide further signal amplification, and 2) secondary antibodiescan be completely replaced with an aptamer designed for a targetmolecule, which is then extended at its 3′ end by TdTase.

7) Signal amplification for DNA-based biomolecular detection. For theultra-sensitive nucleic acid detection such as DNA microarray, targetgenes can be chemically grafted at their 3′ end to apoly(MAA-co-OEGMA)-coated surface and cDNA prepared from a sample thatis hybridized to the target gene can be extended at its 3′ end forsignal amplification (FIG. 8). In addition, molecular beacons withoutfluorophores containing target genes can be chemically grafted at their5′ end (FIG. 8). The binding of cDNA in a sample to the target genecauses unfolding of the molecular beacon so that its 3′ end is no longerhybridized to another part of the beacon and becomes accessible forextension by TdTase. This triggered approach can potentially further cutdown background.

8) Highly adaptable to a variety of detection methods. Signals amplifiedby TdTase can be detected by a variety of methods: 1) direct detectionof the unlabeled DNA tag using the atomic force microscopy (AFM) (FIGS.9 and 10), direct detetcio using SPR spectroscopy (FIG. 11) 3)incorporate radiolabeled nucleotides (3H, 14C, and 32P) directly ingrowing DNA chain for radioactive detection, 4) incorporate stableisotope labeled nucleotides in growing DNA chain and detect bytime-of-flight secondary-ion mass spectrometry (ToF-SIMS) ormatrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS), 5) incorporate fluorophores directly into the DNA chainfollowed by fluorescence detection, 6) incorporate nucleotidescontaining amino acids, to which fluorophores are attached, for moreefficient attachment than with fluorophore-labeled nucleotides, and 7)indirect detection by hybridization of the extended DNA tag with acomplementary full-length DNA strand or short oligonucleotidesfunctionalized with metal nanoparticles, quantum dots, fluorophores, orradionuclides without directly incorporating labeled nucleotides intothe DNA chain.

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The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A biomolecular detector, comprising: (a) a substrate having a surfaceportion; (b) a linking layer on said surface portion; and (c) a polymerlayer formed on said linking layer by the process of surface-initiatedpolymerization of monomeric units thereon, with each of said monomericunits comprising a monomer core group having at least oneprotein-resistant head group coupled thereto, to thereby form a brushmolecule on said surface portion; said brush molecule comprising a stemformed from the polymerization of said monomer core groups, and aplurality of branches formed from said head group projecting from saidstem; and (d) a first member of a specific binding pair coupled to saidbrush molecule.
 2. The detector of claim 1, wherein said member of aspecific binding pair further comprises an extended nucleic acidconjugated thereto, said extended nucleic acid produced by the processof enzymatic extension with terminal transferase.
 3. The detector ofclaim 1, wherein said first member is a first nucleic acid, saiddetector optionally further comprising a second nucleic acid as a secondmember of said binding pair hybridized to said first member.
 4. Thedetector of claim 3, further comprising an extended nucleic acidproduced by the process of enzymatic extension with terminal transferasecoupled to either said first or second nucleic acid.
 5. The detector ofclaim 1, wherein said member of a specific binding pair is a protein orpeptide.
 6. the detector of claim 5, further comprising: a second memberof said specific binding pair coupled to said first member of saidspecific binding pair; an antibody specifically bound to said secondmember to form a sandwich, said antibody having a first nucleic acidconjugated thereto; and an extended nucleotide coupled to said firstnucleotide, said extended nucleic acid produced by the process ofenzymatic extension with terminal transferase.
 7. The detector of claim1, wherein said first member is an antibody.
 8. The detector of claim 1,wherein said surface portion comprises a material selected from thegroup consisting of metals, metal oxides, semiconductors, polymers,silicon, silicon oxide, and composites thereof.
 9. The detector of claim1, wherein said surface portion comprises gold.
 10. The detector ofclaim 1, wherein said linking layer is continuous.
 11. The detector ofclaim 1, wherein said linking layer is patterned.
 12. The detector ofclaim 1, wherein said linking layer is a self-assembled monolayer. 13.The detector of claim 1, wherein said brush molecule is from 5 to 50nanometers in length.
 14. The detector of claim 1, said brush moleculeformed on said surface portion at a density from 40 to 100 milligramsper meter². 15-22. (canceled)
 23. A method of making a biomoleculardetector, said method comprising: (a) providing a substrate having asurface portion; (b) depositing a linking layer on said surface portion;and (c) forming a polymer layer on said linking layer by the process ofsurface-initiated polymerization of monomeric units thereon, with eachof said monomeric units comprising a monomer core group having at leastone protein-resistant head group coupled thereto, to thereby form abrush molecule on said surface portion; said brush molecule comprising astem formed from the polymerization of said monomer core groups, and aplurality of branches formed from said hydrophilic head group projectingfrom said stem; and then (d) covalently coupling a member of a specificbinding pair to said polymer layer.
 24. The method of claim 23, whereinsaid polymer comprises a copolymer of methoxy-terminated OEGMA andhydroxy-terminated OEGMA.
 25. The method of claim 24, further comprisingcoupling a compound having an amine group to said hydroxy group via saidamine group.
 26. The method of claim 25, wherein said copolymer issynthesized on said surface portion by SI-ATRP, wherein said hydroxygroups are converted to COOH groups by reaction with N-hydroxysuccinimide (NHS), and wherein said amine groups are coupled to saidCOOH.
 27. The method of claim 23, wherein said polymer comprises acopolymer of OEGMA and sodium methacrylate, said copolymer having sodiumcarboxylate groups.
 28. The method of claim 27, where said sodiumcarboxylate group is converted to a COOH group by reaction with HCl, anda protein or peptide is coupled to said COOH groups via their N-terminalamine or lysine residues with NHS/EDC coupling.
 29. The method of claim23, wherein said polymer is OEGMA, said OEGMA containing of thioestergroups; and wherein said member of a specific binding pair is coupled tosaid thioester group.
 30. The method of claim 23, wherein said member ofa specific binding pair is a protein or peptide having an N-terminalcysteine, and wherein said coupling step is carried out by reaction withsaid N-terminal cysteine with said thioester by intein protein ligation(IPL) or native chemical ligation.
 31. The method of claim 23, whereinsaid polymer is OEGMA, said OEGMA contains cysteine groups, said memberof a specific binding pair contains a C-terminal thioester, and saidcoupling step is carried out by reaction of said C-terminal cysteinewith said thioester by intein protein ligation (IPL) or native chemicalligation.
 32. The method of claim 23, wherein said surface portioncomprises a material selected from the group consisting of metals, metaloxides, semiconductors, polymers, silicon, silicon oxide, and compositesthereof.
 33. The method of claim 23, wherein said surface portioncomprises gold.
 34. The method of claim 23, wherein said linking layeris continuous.
 35. The method of claim 23, wherein said linking layer ispatterned.
 36. The method of claim 23, wherein said linking layer is aself-assembled monolayer.
 37. The method of claim 23, wherein said brushmolecule is from 5 to 50 nanometers in length.
 38. The method of claim23, said brush molecule formed on said surface portion at a density from40 to 100 milligrams per meter.
 39. The detector of claim 1, whereinsaid brush molecule is produced by surface-initiated atom transferradical polymerization of oligo(ethylene glycol)methyl methacrylate. 40.The detector of claim 1, wherein said head group comprises a kosmotrope.41. The detector of claim 1, wherein said first member of said specificbinding pair is coupled directly to a hydroxyl group in OEGMA by CDIchemistry.
 42. The method of claim 23, wherein said brush molecule isproduced by surface-initiated atom transfer radical polymerization ofoligo(ethylene glycol)methyl methacrylate.